Classical galactosemia and mutations at the galactose‐1‐phosphate uridyl transferase (GALT) gene

Classical galactosemia is caused by a deficiency in activity of the enzyme galactose‐1‐phosphate uridyl transferase (GALT), which, in turn, is caused by mutations at the GALT gene. The disorder exhibits considerable allelic heterogeneity and, at the end of 1998, more than 150 different base changes were recorded in 24 different populations and ethnic groups in 15 countries worldwide. The mutations most frequently cited are Q188R, K285N, S135L, and N314D. Q188R is the most common mutation in European populations or in those predominantly of European descent. Overall, it accounts for 60–70% of mutant chromosomes, but there are significant differences in its relative frequency in individual populations. Individuals homoallelic for Q188R tend to have a severe phenotype and this is in keeping with the virtually complete loss of enzyme activity observed in in vitro expression systems. Globally, K285N is rarer, but in many European populations it can be found on 25–40% of mutant chromosomes. It is invariably associated with a severe phenotype. S135L is found almost exclusively in African Americans. In vitro expression results are discrepant, but some individuals carrying S135L appear to exhibit GALT activity in some tissues. Duarte 1 (or Los Angeles) and Duarte 2 (or Duarte) variants carry the same amino acid substitution, N314D, even though D1 is associated with increased erythrocyte GALT activity and D2 with reduced activity. N314D is in linkage disequilibrium with other base changes that differ on the D1 and D2 alleles. N314D does not impair GALT activity in in vitro expression systems. However, there are differences in the abundance of GALT protein in lymphoblastoid cells lines from D2 and D1 individuals. It is unclear whether the specific molecular changes that distinguish the D1 and D2 alleles account for the different activities. The considerable genetic heterogeneity documented to date undoubtedly contributes to the phenotypic heterogeneity that is observed in galactosemia. The additional effects of nonallelic variation and other constitutional factors on phenotypic variability remain to be elucidated. Hum Mutat 13:417–430, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of mutations in the galactose-1-phosphate uridyltransferase (GALT) gene in 16 Turkish patients with galactosemia, including a novel mutation of F294Y. Mutation in brief no. 235. Online

Classical galactosemia caused by deficiency of galactose-1-phosphate uridyltransferase (GALT) is a severe autosomal recessive disorder. We report here molecular analysis of 16 unrelated Turkish galactosemia index cases without GALT activity. Almost 84% of all mutant alleles were identified in this study. The most common molecular defect observed in the Turkish population was Q188R (replacement of glutamine-188 by arginine) (57%). In order to facilitate the determination of unknown mutations in the entire coding region of GALT, we established an approach based on GALT cDNA synthesis and direct sequencing. We have identified one novel candidate galactosemia mutation, a T-to-A transversion at the codon 294 (F294Y) in exon 9 in addition to previously reported three missense (M142K K285N, A320T), one stop codon (E340X), and one silent (L218L) mutations in galactosemia patients which reflect considerable genetic heterogeneity in the Turkish population.     

Mutation database for the galactose-1-phosphate uridyltransferase (GALT) gene

Classical galactosemia is an autosomal recessive disorder caused by mutations in the galactose-1-phosphate uridyltransferase (GALT) gene. Our group developed a disease-specific database containing all of the reported sequence variants in GALT (Available at: http://arup.utah.edu/database/galactosemia/GALT_welcome.php; Last accessed: 13 April 2007). Currently the database contains a total of 229 sequence variants, of which 196 are mutations (including nine novel mutations identified in our laboratory), 31 polymorphisms in both introns and exons, and two variants of unknown or uncertain significance. All sequence variants have been verified for their position within the GALT gene and named following standard nomenclature. Sequence variants are reported with accompanying information on protein effect, classification of mutation vs. polymorphism, mutation type (when applicable) based on how each was first described in the literature, and accompanying link to pertinent publication. Unpublished variants are described with relevant clinical information that supports their classification as causative of the disease vs. polymorphisms. Other features of this database include disease information, relevant links for galactosemia and literature, reference sequences, ability to query by various criteria, and submit of novel variations to the database. This free online scientific resource was developed with the clinical laboratory in mind to serve as a reference and repository for novel findings that are periodically collected, verified, and updated into the database.     

Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) - deficient mouse model

The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT gene-trapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P=0.02) and longer time-to-pregnancy (P=0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P=0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies.     

Mutation analysis of the GALT gene in Czech and Slovak galactosemia populations: identification of six novel mutations, including a stop codon mutation (X380R)

A study of the galactose-1-phosphate uridyltransferase (GALT) gene from 37 unrelated galactosemia families is reported here. A total of 16 sequence variations in eleven mutated alleles was found. The two most common molecular defects were the mutations Q188R (46.0%) and K285N (25.7%). Six novel mutations in the GALT gene, X380R, Y209S, E340K, L74fsdelCT, Q169K and L256/P257delGCC, were detected. Three mutations, V151A, L195P and R204X that were previously described in other populations, were also found. The mutation X380R, which breaks the stop codon of the GALT gene, causes elongation of the GALT enzyme's protein chain. A deletion of four nucleotides in the 5' promoter region, in a position 116 - 119 nucleotides upstream from the initiate codon (5'UTR-119delGTCA), was revealed in Duarte (D2) alleles, in addition to N314D, IVS4nt-27g-->c, IVS5nt+62g-->a, and IVS5nt-24g-->a. An unusual molecular genotype was observed on 2 types of classical galactosemia alleles, with six variations from the normal nucleotide sequence presented in cis (mutation V151A or E340K plus five Duarte (D2) characteristic variations). In summary, galactosemia is a heterogeneous disorder at the molecular level, and mutation N314D, appears to be an ancient genetic variant of the GALT gene. Hum Mutat 15:206, 2000.     

Identification of novel C253Y missense and Y864X nonsense mutations in the insulin receptor gene in type A insulin-resistant patients

Mutations in the insulin receptor gene have been reported in cases of type A insulin resistance. We report herein two cases of type A insulin resistance, which involve some novel mutations. Case 1 is a heterozygote of the C253Y missense mutation and case 2 is a heterozygote of the Y864X nonsense mutation. In the C253Y missense mutation in exon 3, a cysteine residue is replaced with tyrosine in the cysteine-rich domain of the alpha subunit. The Y864X in exon 13 results in a truncated receptor, which is devoid of most of the beta subunit. This mutant receptor could not be expressed on a cell membrane since the transmembrane domain is missing. Other significant mutations were not found for the entire coding regions and splice/donor sites.     

Bruton tyrosine kinase gene mutations in Turkish patients with presumed X‐linked agammaglobulinemia

X‐linked agammglobulinemia (XLA) is a ptototypical humoral immunodeficiency caused by mutations in the gene coding for Bruton tyrosine kinase (BTK). The genetic defect in XLA impairs early B cell development resulting in marked reduction of mature B cells in the blood. Studies from different countries have demonstrated that approximately 90% of males with presumed XLA bear mutations in BTK. In this study, we report for the first time the occurrence of BTK mutations in Turkey. We performed mutational analysis of the BTK gene in 16 Turkish male patients from 13 separate families with presumed XLA based on abnormally low peripheral blood B‐cell numbers (lt; 1%), hypogammaglobulinemia, and recurrent bacterial infections. We found that in nine of the 13 families (69%) a Btk mutation caused XLA. Two of the mutations were previously described, but seven novel mutations were identified: two missense (Y39C, G584R), one nonsense (Q343X), and 4 deletions (1800‐1821del, 1843‐1847del, 1288‐1292del, 291del) resulting in frameshift and premature stop codon. By contrast, no mutations in the BTK gene were identified in the other 4 families. A consanguinity in three of these families raises the possibility that mutations in other autosomal genes which affect early B cell development may contribute to their phenotype resembling XLA. Hum Mutat 18:356, 2001. © 2001 Wiley‐Liss, Inc.     

Molecular analysis of Turkish mucopolysaccharidosis IVA (Morquio A) patients: identification of novel mutations in the N-acetylgalactosamine-6-sulfate sulfatase (GALNS) gene

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disorder caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS; EC 3.1.6.4). The deficiency of N-acetylgalactosamine-6-sulfate sulfatase leads to lysosomal accumulation of undegraded glycosaminoglycans, keratan sulfate and chondroitin-6-sulfate. Mutation screening of the GALNS gene was performed by SSCP and direct sequence analyses using genomic DNA samples from 10 Morquio A patients. By nonradioactive SSCP screening, 6 different gene mutations and 2 polymorphisms were identified in 10 severely affected MPS IVA patients. Five of the mutations and one of the polymorphisms are novel. The vast majority of the gene alterations were found to be single nucleotide deletions (389delG, 929delG, and 763delT) or insertions (1232-1233insT). The other two mutations were one previously identified missense mutation (Q473X) and one novel nonsense (P179S) mutation. Together they account for 95% of the disease alleles of the patients investigated. Beside mutations, one previously identified E477 polymorphism and one novel W520 polymorphism were found among Turkish MPS IVA patients.     

Transglutaminase‐1 gene mutations in autosomal recessive congenital ichthyosis: Summary of mutations (including 23 novel) and modeling of TGase‐1

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of rare cornification diseases. Germline mutations in TGM1 are the most common cause of ARCI in the United States. TGM1 encodes for the TGase‐1 enzyme that functions in the formation of the cornified cell envelope. Structurally defective or attenuated cornified cell envelop have been shown in epidermal scales and appendages of ARCI patients with TGM1 mutations. We review the clinical manifestations as well as the molecular genetics of ARCI. In addition, we characterized 115 TGM1 mutations reported in 234 patients from diverse racial and ethnic backgrounds (Caucasion Americans, Norwegians, Swedish, Finnish, German, Swiss, French, Italian, Dutch, Portuguese, Hispanics, Iranian, Tunisian, Moroccan, Egyptian, Afghani, Hungarian, African Americans, Korean, Japanese and South African). We report 23 novel mutations: 71 (62%) missense; 20 (17%) nonsense; 9 (8%) deletion; 8 (7%) splice‐site, and 7 (6%) insertion. The c.877‐2A>G was the most commonly reported TGM1 mutation accounting for 34% (147 of 435) of all TGM1 mutant alleles reported to date. It had been shown that this mutation is common among North American and Norwegian patients due to a founder effect. Thirty‐one percent (36 of 115) of all mutations and 41% (29 of 71) of missense mutations occurred in arginine residues in TGase‐1. Forty‐nine percent (35 of 71) of missense mutations were within CpG dinucleotides, and 74% (26/35) of these mutations were C>T or G>A transitions. We constructed a model of human TGase‐1 and showed that all mutated arginines that reside in the two beta‐barrel domains and two (R142 and R143) in the beta‐sandwich are located at domain interfaces. In conclusion, this study expands the TGM1 mutation spectrum and summarizes the current knowledge of TGM1 mutations. The high frequency of mutated arginine codons in TGM1 may be due to the deamination of 5′ methylated CpG dinucleotides. Hum Mutat 0, 1–12, 2009. © 2009 Wiley‐Liss, Inc.     

Six novel β‐galactosidase gene mutations in Brazilian patients with GM1‐gangliosidosis

GM1‐gangliosidosis is a lysosomal storage disease caused by a deficiency of acid β‐galactosidase. Three clinical forms are recognized—infantile, juvenile, and adult—based on age of onset and severity of the symptoms. We have performed molecular analysis of a large cohort of GM1 patients (19 Brazilian and one Uruguayan), using nonradioactive single‐strand conformation polymorphism (SSCP) and restriction enzyme analysis of genomic DNA. Six novel mutations (R121S, V240M, D491N, 638–641insT, 895–896insC, 1622–1627insG) and two previously described point mutations (R59H, R208C) were identified. Together they accounted for 90% of the disease alleles of the patients. Two mutations, 1622–1627insG and R59H, were present in 18 of 20 patients. In addition, four polymorphisms (L10P, L12L, R521C, S532G) were identified. All cases reported are infantile GM1 gangliosidosis. This report constitutes the most comprehensive molecular study to date of this disorder in infantile patients. Since GM1‐gangliosidosis is the most common lysosomal storage disorder in Southern Brazil, molecular diagnosis will be important for genetic counseling, carrier detection and prenatal diagnosis in index families. Hum Mutat 13:401–409, 1999. © 1999 Wiley‐Liss, Inc.     

A novel missense mutation (402C → T) in exon 1 in the EDA gene in a family with X-linked hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasia (EDA), or Christ-Siemens-Touraine syndrome, is clinically characterized by hypohidrosis, hypoodontia and hypotrichosis. The X-linked form of the disease has been mapped to Xq12-q13.1, and a gene from this region has recently been cloned. This gene encodes a predicted transmembrane protein of 135 amino acids, which was found to be expressed in keratinocytes, hair follicles, and sweat glands. A variety of rearrangements in this gene have been found in patients with hypohidrotic ectodermal dysplasia.
We have screened the probands from nine unrelated Danish families with hypohidrotic ectodermal dysplasia for mutation in exon 1 of the EDA-gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). In one large kindred we identified a novel missense mutation (402C → T), which changes a histidine to tyrosine at position 54 in the protein. This mutation cosegregates with the disease in the family and is the first mutation described which affects the predicted transmembrane, hydrophobic domain of the protein.     

Identification of 16 sulfamidase gene mutations including the common R74C in patients with mucopolysaccharidosis type IIIA (Sanfilippo A)

Mucopolysaccharidosis type IIIA (MPS IIIA or Sanfilippo A disease) is a storage disorder caused by deficiency of the lysosomal enzyme sulfamidase. Mutation screening, using SSCP/heteroduplex analyses on cDNA and genomic DNA fragments, was performed in a group of 42 European patients. Sixteen of the 17 different gene mutations characterized have not been previously described. The spectrum of gene lesions consists of two 1-bp deletions (1091delC, 1093delG), an 18-bp duplication (421ins18), a splice site mutation (IVS2-2A→G), and 13 different missense point mutations. As in other lysosomal storage disorders, the phenotypic heterogeneity is associated with a considerable genetic heterogeneity. The missense mutation R74C, which alters an evolutionary conserved amino acid in the active site of the enzyme, was found on 56% of alleles of 16 Polish patients, whereas it was less frequent among German patients (21% of disease alleles). R245H, a previously reported common mutation, represents 35% of disease alleles in German patients, but only 3% in Polish patients. As the combined frequency of the common mutations (R74C and R245H) in German and Polish populations exceeds 55%, screening for these two mutations will assist molecular genetic diagnosis of MPS IIIA and allow heterozygote testing in these populations. Hum Mutat 10:479–485, 1997. © 1997 Wiley-Liss, Inc.     

Identification of a novel point mutation (S65T) in α-galactosidase A gene in Chinese patients with Fabry disease

Fabry disease is an X-linked inborn error of sphingolipid catabolism resulting from deficient enzyme activity of α-galactosidase A. The molecular defects of human α-galactosidase A gene causing Fabry disease have been characterized, including gene rearrangement and point mutations, which show the genetic heterogeneity in Fabry disease. To characterize the molecular defects of these patients, each exon of α-galactosidase A gene including intron-exon junctions were PCR amplified using biotin-labelled primer and sequenced using magnetic beads solid-phase sequencing. A G to C transversion was identified in the last nucleotide of exon 1 in two unrelated Chinese patients. This mutation obliterates an EcoN1 restriction site. Family studies show close linkage with the affected family members. Screening of 100 alleles (22 males, 39 females) of unrelated normal Chinese can not find this mutation. This mutation not only changes the amino acid from serine to threonine, but also likely cause splicing defects. To our knowledge, this is the first report of mutation in Chinese patients with Fabry disease, and a novel mutation causing Fabry disease not reported in literature previously. Hum Mutat 11:328–330, 1998. © 1998 Wiley-Liss, Inc.     

Treatment for hereditary angioedema with normal C1‐INH and specific mutations in the F12 gene (HAE‐FXII)

Hereditary angioedema with normal C1 esterase inhibitor and mutations in the F12 gene (HAE‐FXII) is associated with skin swellings, abdominal pain attacks, and the risk of asphyxiation due to upper airway obstruction. It occurs nearly exclusively in women. We report our experience treating HAE‐FXII with discontinuation of potential trigger factors and drug therapies. The study included 72 patients with HAE‐FXII. Potential triggers included estrogen‐containing oral contraceptives (eOC), hormonal replacement therapy, or angiotensin‐converting enzyme inhibitors. Drug treatment comprised plasma‐derived C1 inhibitor (pdC1‐INH) for acute swelling attacks and progestins, tranexamic acid, and danazol for the prevention of attacks. Discontinuation of eOC was effective in 25 (89.3%) of 28 women and led to a reduction in the number of attacks (about 90%). After ending hormonal replacement therapy, three of eight women became symptom‐free. Three women with exacerbation of HAE‐FXII during intake of quinapril or enalapril had no further HAE‐FXII attacks after discontinuation of those drugs. Eleven women were treated with pdC1‐INH for 143 facial attacks. The duration of the treated facial attacks (mean: 26.6 h; SD: 10.1 h) was significantly shorter than that of the previous 88 untreated facial attacks in the same women (mean: 64.1 h; SD: 28.0 h; P < 0.01). The mean reduction in attack frequency was 99.8% under progestins after discontinuing eOC (16 women), 93.8% under tranexamic acid (four women), and 100% under danazol (three women). For patients with HAE‐FXII, various treatment options are available which completely or at least partially reduce the number or duration of attacks.     

Identification of a novel R21X mutation in the liver‐type arginase gene (ARG1) in four Portuguese patients with argininemia

Argininemia is a rare autossomal recessive disorder caused by deficiency in the cytosolic liver‐type arginase enzyme (L‐arginine urea‐hydrolase; E.C. 3.5.3.1). In order to investigate the molecular basis for argininemia in four unrelated Portuguese patients (two from northern Portugal and two from Madeira Island) we performed a DNA sequence analysis of all the exons and exon/intron boundaries of the liver‐type arginase gene (ARG1). All patients were found to be homozygous for a newly identified C ‐>T transition in codon 21 (exon 2) substituting arginine for a premature stop codon (R21X: CGA to TGA) and generating a NlaIII restriction site. Restriction digestion following PCR amplification of ARG1 exon 2 confirmed the presence of the mutation. Hum Mutat 14:355–356, 1999. © 1999 Wiley‐Liss, Inc.     

Acute intermittent porphyria: Characterization of two novel mutations in the porphobilinogen deaminase gene,one amino acid deletion (453‐455delAGC) and one splicing aceptor site mutation (IVS8‐1G>T)

A partial deficiency of Porphobilinogen deaminase (PBG‐D) is responsible for acute intermittent porphyria (AIP). AIP is inherited in an autosomal dominant fashion, and the prevalence in the Argentinean population is about 1:125,000. Here, two new mutations and three previously reported were found in the PBG‐D gene in 12 Argentinean AIP patients corresponding to 5 different families. To screen for AIP mutations in symptomatic patients, genomic DNA isolated was amplified in 2 Multiplex PCR reactions, then all coding exons and flanking intronic regions were sequenced. The new mutations are 453‐455delAGC in exon 9 which results in the loss of an alanine residue at position 152, and one new point mutation in the splicing aceptor site in the last position of intron 8 (IVS8‐1G>T) which leds to a 15 bp deletion because a cryptic site (first AG upstream) is used. Both mutations produce amino acid deletion without frameshift effect. To further characterize the 453‐455delAGC mutation, the pKK‐PBGD construct for the mutant allele was expressed in E. coli, the enzymatic activity of the recombinant protein was 1.3% of the mean level expressed by the normal allele. Finally, three missense mutations, previously reported, were identified in three unrelated families. Hum Mutat 14:355, 1999. © 1999 Wiley‐Liss, Inc.